Oridonin ameliorates renal fibrosis in diabetic nephropathy by inhibiting the Wnt/ß-catenin signaling pathway.

Diabetic nephropathy (DN) is one of the most serious and frequent complications among diabetes patients and presently constitutes vast the cases of end-stage renal disease worldwide. Tubulointerstitial fibrosis is a crucial factor related to the occurrence and progression of DN. Oridonin (Ori) is a diterpenoid derived from rubescens that has diverse pharmacological properties. Our previous study showed that Ori can protect against DN by decreasing the inflammatory response. However, whether Ori can alleviate renal fibrosis in DN remains unknown. Here, we investigated the mechanism through which Ori affects the Wnt/ß-catenin signaling pathway in diabetic rats and human proximal tubular epithelial cells (HK-2) exposed to high glucose (HG) levels. Our results revealed that Ori treatment markedly decreased urinary protein excretion levels, improved renal function and alleviated renal fibrosis in diabetic rats. In vitro, HG treatment increased the migration of HK-2 cells while reducing their viability and proliferation rate, and treatment with Ori reversed these changes. Additionally, the knockdown of ß-catenin arrested cell migration and reduced the expression levels of Wnt/ß-catenin signaling-related molecules (Wnt4, p-GSK3ß and ß-catenin) and fibrosis-related molecules (α-smooth muscle actin, collagen I and fibronectin), and Ori treatment exerted an effect similar to that observed after the knockdown of ß-catenin. Furthermore, the combination of Ori treatment and ß-catenin downregulation exerted more pronounced biological effects than treatment alone. These findings may provide the first line of evidence showing that Ori alleviates fibrosis in DN by inhibiting the Wnt/ß-catenin signaling pathway and thereby reveal a novel therapeutic avenue for treating tubulointerstitial fibrosis.

Oridonin represses lung cancer cell proliferation and migration by modulating AFAP1-AS1/IGF2BP1.

Oridonin belongs to a small molecule from the Chinese herb Rabdosia rubescens with potent anticancer activity. In spite of the lncRNA AFAP1-AS1 has been proven to exert promoting function in lung cancer, its relationship with oridonin in lung cancer is obscure. Therefore, our study planned to explore the potential of oridonin in lung cancer as well as unveil the regulatory mechanism of oridonin on AFAP1-AS1 in lung cancer cells. In the present study, oridonin inhibited lung cancer cell proliferation, migration, as well as invasion, as evidenced by MTT, wound healing, as well as transwell assays. Besides, we observed that oridonin could downregulate AFAP1-AS1 expression, and overexpressed AFAP1-AS1 could reverse the repressive effects of oridonin on lung cancer cell proliferation, migration, as well as invasion. More importantly, we found that AFAP1-AS1 could bind to IGF2BP1 through starBase prediction and RIP assay. The expression level of IGF2BP1 was also reduced by oridonin treatment but reversed after AFAP1-AS1 overexpression. Additionally, we proved that overexpressed IGF2BP1 could reverse the repressive impacts of oridonin on lung cancer cell proliferation, migration, as well as invasion. Further, in vivo experiments validated the repressive role of oridonin on tumor growth of lung cancer. Together, oridonin inhibits lung cancer cell proliferation as well as migration by modulating AFAP1-AS1/IGF2BP1, and AFAP1-AS1/IGF2BP1 possesses the potential to be a promising therapy targeting for lung cancer, especially in oridonin treatment.

Oridonin, a small molecule inhibitor of cancer stem cell with potent cytotoxicity and differentiation potential.

Cancer stem cells (CSCs) drive malignant tumor progression, recurrence, and metastasis with unique characteristics, including self-renewal and resistance to conventional treatments. Conventional differentiation inducers, although promising, have limited cytotoxicity and may inadvertently enhance CSC stemness. To address these challenges, ongoing efforts are dedicated to developing strategies that can effectively combine both cytotoxicity and differentiation-inducing effects. In this study, we introduce oridonin (Ori), a small molecule with dual differentiation-inducing and cytotoxicity properties capable of eliminating tumor CSCs. We isolated CSCs in B16F10 cells using the Hoechst side population method and assessed the differentiation effect of Ori. Ori's differentiation-inducing effect was further evaluated using human acute promyelocytic leukemia. The cytotoxic potential of Ori against MCF-7 and B16F10 cell lines was assessed through various methods. In vivo anti-tumor and anti-CSC efficacy of Ori was investigated using mouse melanoma and CSCs melanoma models. Safety evaluation included zebrafish embryotoxicity and mouse acute toxicity experiments. As a result, Ori effectively dismantles tumorspheres, inhibits proliferation, and reduces the expression of CSC-specific markers. It induces significant differentiation, especially in the case of NB4. Additionally, Ori upregulates TP53 expression, mitigates the hypoxic tumor microenvironment, suppresses stemness, and inhibits PD-L1 expression, prompting a robust anti-cancer immune response. Ori demonstrates pronounced cytotoxicity, inducing notable pro-apoptotic effects on B16F10 and MCF-7 cells, with specific triggering of mitochondrial apoptosis. Importantly, Ori maintains a commendable biosafety record. The dual-action prowess of Ori not only induces the differentiation of CSCs but also dispatches differentiated and residual tumor cells, effectively thwarting the relentless march of tumor progression.

Properties, extraction and purification technologies of Stevia rebaudiana steviol glycosides: A review.

For health and safety reasons, the search for green, healthy, and low-calorie sweeteners with good taste has become the demand of many consumers. Furthermore, the need for sugar substitutes of natural origin has increased dramatically. In this review, we briefly discussed the safety and health benefits of stevia sweeteners and enumerated some examples of physiological functions of steviol glycosides (SGs), such as anti-inflammatory, anti-obesity, antihypertensive, anti-diabetes, and anticaries, citing various evidence related to their application in the food industry. The latest advances in emerging technologies for extracting and purifying SGs and the process variables and operational strategies were discussed. The impact of the extraction methods and their comparison against the conventional techniques have also been demonstrated. These technologies use minimal energy solvents and simplify subsequent purification stages, making viable alternatives suitable for a possible industrial application. Furthermore, we also elucidated the potential for advancing and applying the natural sweeteners SGs.

Suppression of PERK/eIF2α/CHOP pathway enhances oridonin-induced apoptosis by inhibiting autophagy in Small-Cell lung cancer cells.

Chinese herbs have been used to treat small-cell lung cancer (SCLC) due to their low toxicity and significant efficacy. This study focused on oridonin, a natural compound extracted from Rabdosia rubescens, and aimed to investigate its potential antitumor activity on SCLC and to evaluate the synergistic effect of combining oridonin with other small molecules. In this study, oridonin exhibited a dual effect. At lower concentrations, it suppressed the cell viability of SCLC cells (H1688 and H446). At high concentrations, oridonin induced SCLC cell apoptosis, damaged HBE cells in vitro and compromised the function of the liver and heart in vivo. The lower concentration of oridonin induced autophagy by enhancing the expression of p62 and the LC3B-II/LC3B-I ratio. This phenomenon might be associated with the activation of the protein kinase RNA-like ER kinase (PERK)/eukaryotic initiation factor 2 alpha (eIF2α)/growth arrest and DNA damage-inducible gene 153 (CHOP/GAD153) pathway. Therefore, the combined effect of oridonin with GSK2606414 or 3- methyladenine increased apoptosis in SCLC cells and reduced tumor growth. A similar phenomenon was observed after oridonin was combined with p62 or CHOP RNA interference treatment. Simultaneously, the combination of oridonin and GSK2606414 exhibited therapeutic efficacy without manifesting adverse effects. Our findings suggest that oridonin at lower concentrations can induce autophagy by activating the PERK/eIF2α/CHOP signaling pathway. The inhibition of the PERK/eIF2α/CHOP pathway could enhance oridonin therapeutic responses by triggering apoptosis. The novel therapeutic approach of combining oridonin with a PERK inhibitor is promising as a strategy for the treatment of SCLC.

Oridonin exerts anticonvulsant profile and neuroprotective activity in epileptic mice by inhibiting NLRP3-mediated pyroptosis.

BACKGROUND: Epilepsy is a chronic disabling disease poorly controlled by available antiseizure medications. Oridonin, a bioactive alkaloid with anti-inflammatory properties and neuroprotective effects, can inhibit the increased excitability of neurons caused by glutamate accumulation at the cellular level. However, whether oridonin affects neuronal excitability and whether it has antiepileptic potential has not been reported in animal models or clinical studies. METHOD: Pentylenetetrazol was injected into mice to create a model of chronic epilepsy. Seizure severity was assessed using the Racine scale, and the duration and latency of seizures were observed. Abnormal neuronal discharge was detected using electroencephalography, and neuronal excitability was assessed using calcium imaging. Damage to hippocampal neurons was evaluated using Hematoxylin-Eosin and Nissl staining. The expression of the NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome and other pyroptosis-related proteins was determined using western blotting and immunofluorescence. A neuronal pyroptosis model was established using the supernatant of BV2 cells treated with lipopolysaccharide and adenosine triphosphate to stimulate hippocampal neurons. RESULTS: Oridonin (1 and 5 mg/kg) reduced neuronal damage, increased the latency of seizures, and shortened the duration of fully kindled seizures in chronic epilepsy model mice. Oridonin decreased abnormal discharge during epileptic episodes and suppressed increased neuronal excitability. In vitro experiments showed that oridonin alleviated pyroptosis in hippocampal HT22 neurons. CONCLUSION: Oridonin exerts neuroprotective effects by inhibiting pyroptosis through the NLRP3/caspase-1 pathway in chronic epilepsy model mice. It also reduces pyroptosis in hippocampal neurons in vitro, suggesting its potential as a therapy for epilepsy.

Glutathione-depleting Liposome Adjuvant for Augmenting the Efficacy of a Glutathione Covalent Inhibitor Oridonin for Acute Myeloid Leukemia Therapy.

BACKGROUND: Discrepancies in the utilization of reactive oxygen species (ROS) between cancer cells and their normal counterparts constitute a pivotal juncture for the precise treatment of cancer, delineating a noteworthy trajectory in the field of targeted therapies. This phenomenon is particularly conspicuous in the domain of nano-drug precision treatment. Despite substantial strides in employing nanoparticles to disrupt ROS for cancer therapy, current strategies continue to grapple with challenges pertaining to efficacy and specificity. One of the primary hurdles lies in the elevated levels of intracellular glutathione (GSH). Presently, predominant methods to mitigate intracellular GSH involve inhibiting its synthesis or promoting GSH efflux. However, a conspicuous gap remains in the absence of a strategy capable of directly and efficiently clearing GSH. METHODS: We initially elucidated the chemical mechanism underpinning oridonin, a diminutive pharmacological agent demonstrated to perturb reactive oxygen species, through its covalent interaction with glutathione. Subsequently, we employed the incorporation of maleimide-liposomes, renowned for their capacity to disrupt the ROS delivery system, to ameliorate the drug's water solubility and pharmacokinetics, thereby enhancing its ROS-disruptive efficacy. In a pursuit to further refine the targeting for acute myeloid leukemia (AML), we harnessed the maleic imide and thiol reaction mechanism, facilitating the coupling of Toll-like receptor 2 (TLR2) peptides to the liposomes' surface via maleic imide. This strategic approach offers a novel method for the precise removal of GSH, and its enhancement endeavors are directed towards fortifying the precision and efficacy of the drug's impact on AML targets. RESULTS: We demonstrated that this peptide-liposome-small molecule machinery targets AML and consequently induces cell apoptosis both in vitro and in vivo through three disparate mechanisms: (I) Oridonin, as a Michael acceptor molecule, inhibits GSH function through covalent bonding, triggering an initial imbalance of oxidative stress. (II) Maleimide further induces GSH exhaustion, aggravating redox imbalance as a complementary augment with oridonin. (III) Peptide targets TLR2, enhances the directivity and enrichment of oridonin within AML cells. CONCLUSION: The rationally designed nanocomplex provides a ROS drug enhancement and targeted delivery platform, representing a potential solution by disrupting redox balance for AML therapy.

[Functional characterization of ent-kaurane-type diterpenoid synthases from Stellera chamaejasme].

Sequential catalysis by ent-copalyl diphosphate(CPS) and ent-kaurene synthase(KS) is a critical step for plants to initiate the biosynthesis of gibberellin with geranylgeranyl pyrophosphate(GGPP) as the substrate. This study mined the transcriptome data of Stellera chamaejasme and cloned two key diterpene synthase genes, SchCPS and SchKS, involved in the gibberellin pathway. The two genes had the complete open reading frames of 2 595 bp and 1 701 bp, encoding two hydrophilic proteins composed of 864 and 566 amino acid residues and with the relative molecular mass of 97.9 kDa and 64.6 kDa and the theoretical isoelectric points of 5.61 and 6.12, respectively. Sequence comparison and phylogenetic tree showed that SchCPS contained LHS, PNV, and DxDD motifs conserved in the CPS family and was categorized in the TPS-c subfamily, while SchKS contained DDxxD, NSE/DTE and PIx motifs conserved in the KS family and was categorized in the TPS-e subfamily. Functional validation showed that SchCPS catalyzed the protonation and cyclization of GGPP to ent-CPP, while SchKS acted on ent-CPP dephosphorylation and re-cyclization to ent-kaurene. In this study, the full-length sequences of SchCPS and SchKS were cloned and functionally verified for the first time, which not only enriched the existing CPS and KS gene libraries but also laid a foundation for the cloning and biosynthesis pathway analysis of more genes involved in the synthesis of active components in S. chamaejasme.

Stevioside Ameliorates Palmitic Acid-Induced Abnormal Glucose Uptake via the PDK4/AMPK/TBC1D1 Pathway in C2C12 Myotubes.

BACKGROUND: Stevioside (SV) with minimal calories is widely used as a natural sweetener in beverages due to its high sweetness and safety. However, the effects of SV on glucose uptake and the pyruvate dehydrogenase kinase isoenzyme (PDK4) as an important protein in the regulation of glucose metabolism, remain largely unexplored. In this study, we used C2C12 skeletal muscle cells that was induced by palmitic acid (PA) to assess the effects and mechanisms of SV on glucose uptake and PDK4. METHODS: The glucose uptake of C2C12 cells was determined by 2-NBDG; expression of the Pdk4 gene was measured by quantitative real-time PCR; and expression of the proteins PDK4, p-AMPK, TBC1D1 and GLUT4 was assessed by Western blotting. RESULTS: In PA-induced C2C12 myotubes, SV could significantly promote cellular glucose uptake by decreasing PDK4 levels and increasing p-AMPK and TBC1D1 levels. SV could promote the translocation of GLUT4 from the cytoplasm to the cell membrane in cells. Moreover, in Pdk4-overexpressing C2C12 myotubes, SV decreased the level of PDK4 and increased the levels of p-AMPK and TBC1D1. CONCLUSION: SV was found to ameliorate PA-induced abnormal glucose uptake via the PDK4/AMPK/TBC1D1 pathway in C2C12 myotubes. Although these results warranted further investigation for validation, they may provide some evidence of SV as a safe natural sweetener for its use in sugar-free beverages to prevent and control T2DM.

Efficient Conversion of Stevioside to Rebaudioside M in Saccharomyces cerevisiae by a Engineering Hydrolase System and Prolonging the Growth Cycle.

Rebaudioside (Reb) M is an important sweetener with high sweetness, but its low content in Stevia rebaudiana and low catalytic capacity of the glycosyltransferases in heterologous microorganisms limit its production. In order to improve the catalytic efficiency of the conversion of stevioside to Reb M by Saccharomyces cerevisiae, several key issues must be resolved including knocking out endogenous hydrolases, enhancing glycosylation, and extending the enzyme catalytic process. Herein, endogenous glycosyl hydrolase SCW2 was knocked out in S. cerevisiae. The glycosylation process was enhanced by screening glycosyltransferases, and UGT91D2 from S. rebaudiana was identified as the optimum glycosyltransferase. The UDP-glucose supply was enhanced by overexpressing UGP1, and co-expressing UGT91D2 and UGT76G1 achieved efficient conversion of stevioside to Reb M. In order to extend the catalytic process, the silencing information regulator 2 (SIR2) which can prolong the growth cycle of S. cerevisiae was introduced. Finally, combining these modifications produced 12.5 g/L Reb M and the yield reached 77.9% in a 5 L bioreactor with 10.0 g/L stevioside, the highest titer from steviol glycosides to Reb M reported to date. The engineered strain could facilitate the industrial production of Reb M, and the strategies provide references for the production of steviol glycosides.

Identifying Potential Sources of Phthalate Contamination in the Leaves of Stevia Rebaudiana (Bertoni) and the Development of Removal Technology.

Steviosides extracted from the leaves of the plant Stevia rebaudiana are increasingly used in the food industry as natural low-calorie sweeteners. Phthalates in food are often assumed to arise from food containers or packaging materials. Here, experiments were carried out to identify the potential sources of DMP, DBP, DIBP, and DEHP in the leaves of stevioside through investigation of their content in native stevioside tissues, soils, and associated agronomic materials. The results show that phthalate contamination was present in all the samples tested, and the influence of regional factors at the provincial level on the content of plasticizers in stevia leaves was not significant. Phthalates in stevia leaves can be absorbed into the plant body through leaves and roots. Using resin removal, the phthalate content in stevioside glycosides was reduced to less than 0.05 ppm, and some indicators were far lower than the limit standard in EU food.

Design, Synthesis, and In Vivo Evaluation of Isosteviol Derivatives as New SIRT3 Activators with Highly Potent Cardioprotective Effects.

Cardiovascular diseases (CVDs) persist as the predominant cause of mortality, urging the exploration of innovative pharmaceuticals. Mitochondrial dysfunction stands as a pivotal contributor to CVDs development. Sirtuin 3 (SIRT3), a prominent mitochondrial deacetylase known for its crucial role in protecting mitochondria against damage and dysfunction, has emerged as a promising therapeutic target for CVDs treatment. Utilizing isosteviol, a natural ent-beyerene diterpenoid, 24 derivatives were synthesized and evaluated in vivo using a zebrafish model, establishing a deduced structure-activity relationship. Among these, derivative 5v exhibited significant efficacy in doxorubicin-induced cardiomyopathy in zebrafish and murine models. Subsequent investigations revealed that 5v selectively elevated SIRT3 expression, leading to the upregulation of SOD2 and OPA1 expression, effectively preventing mitochondrial dysfunction, mitigating oxidative stress, and preserving cardiomyocyte viability. As a novel structural class of SIRT3 activators with robust therapeutic effects, 5v emerges as a promising candidate for further drug development.

Synthetic Derivatives of Natural ent-Kaurane Atractyligenin Disclose Anticancer Properties in Colon Cancer Cells, Triggering Apoptotic Cell Demise.

The antitumor activity of different ent-kaurane diterpenes has been extensively studied. Several investigations have demonstrated the excellent antitumor activity of synthetic derivatives of the diterpene atractyligenin. In this research, a series of new synthetic amides and their 15,19-di-oxo analogues obtained from atractyligenin by modifying the C-2, C-15, and C-19 positions were designed in order to dispose of a set of derivatives with different substitutions at the amidic nitrogen. Using different concentrations of the obtained compounds (10-300 µM) a reduction in cell viability of HCT116 colon cancer cells was observed at 48 h of treatment. All the di-oxidized compounds were more effective than their alcoholic precursors. The di-oxidized compounds had already reduced the viability of two colon cancer cells (HCT116 and Caco-2) at 24 h when used at low doses (2.5-15 µM), while they turned out to be poorly effective in differentiated Caco-2 cells, a model of polarized enterocytes. The data reported here provide evidence that di-oxidized compounds induced apoptotic cell death, as demonstrated by the appearance of condensed and fragmented DNA in treated cells, as well as the activation of caspase-3 and fragmentation of its target PARP-1.

Oridonin promotes RSL3-induced ferroptosis in breast cancer cells by regulating the oxidative stress signaling pathway JNK/Nrf2/HO-1.

The incidence and mortality of breast cancer, the most common malignant tumor among women in the world, are increasing year by year, which greatly threatens women's health. Ferroptosis is an iron and lipid reactive oxygen species (ROS)-dependent process, a novel form of cell death that is distinct from apoptosis and is closely related to the progression of breast cancer. Inducing the occurrence of ferroptosis in tumor cells can effectively block its malignant progress in vivo. Oridonin (ORI), the primary active ingredient extracted from the Chinese herbal medicine Rabdosia rubescens, has been shown to cause glutathione depletion and directly inhibit glutathione peroxidase 4 induced cell death by ferroptosis, but its mechanism of action in breast cancer remains inadequately elucidated. Therefore, we further investigated whether ORI could promote RSL3-induced ferroptosis in breast cancer cells by regulating the oxidative stress pathway JNK/Nrf2/HO-1. In our study, we assessed cell survival of RSL3 and ORI treatment by MTT assay, and found that co-treatment with RSL3 and ORI inhibited cell proliferation, as evidenced by the cloning assay. To investigate the ability of ORI to promote RSL3-induced ferroptosis in breast cancer cells, we measured levels of ROS, malondialdehyde, glutathione, superoxide dismutase, and Fe2+ content. Lipid peroxidation, ROS, and mitochondrial membrane potential levels induced by co-treatment of ORI with RSL3 were reversed by ferrostatin-1, further confirming that the cell death induced by RSL3 and ORI was ferroptosis rather than other programmed cell death modes. Moreover, RSL3 and ORI co-treatment regulated the JNK/Nrf2/HO-1 axis, as demonstrated by western blotting and target activator validation. Our results showed that ORI could enhance the inhibitory effect of RSL3 on breast cancer cells viability via the induction of ferroptosis. Mechanistically, it potentiated RSL3-induced ferroptosis in breast cancer cells by activating the JNK/Nrf2/HO-1 axis. This study provides a theoretical basis for the application of ORI based on the mechanism of ferroptosis, and provides potential natural drug candidates for cancer prevention and treatment.

Stevioside mitigates lead toxicity in thinlip mullet juveniles: Impacts on growth, metabolism, and immune function.

This study investigates the impact of varying concentrations of stevioside in the presence of lead (Pb) exposure on multiple aspects of thinlip mullet (Liza ramada) juveniles. Over 60 days, a total of 540 juvenile L. ramada with an initial weight of 3.5 ± 0.13 g were evenly distributed into six groups, each consisting of three replicates. The experimental diet consisted of varying levels of stevioside (150, 250, 350, and 450 mg/kg diet), with a consistent concentration of lead (Pb) set at 100 µg/kg diet. Stevioside demonstrated a positive influence on growth parameters, with the 450 mg/kg +Pb treatment showing the highest values. Biochemical parameters remained stable, but lead-exposed fish without stevioside displayed signs of potential liver damage and metabolic issues. Stevioside supplementation, especially at higher doses (≥250 mg/kg), reversed these negative effects, restoring biochemical markers to healthy control levels. Lead exposure significantly suppressed antioxidant enzyme activities, but co-administration of stevioside exhibited a dose-dependent protective effect, with 250, 350, and 450 mg/kg groups showing activities comparable to the healthy control. Lead-exposed fish without stevioside demonstrated attenuation of the immune response, but stevioside supplementation reversed these effects, particularly at ≥250 mg/kg. Stev (≥250 mg/kg) reduced IL-1ß and hepcidin expression, contrasting dose-dependent upregulation in lower dosages and lead-only group. Histological examinations of the intestine and liver supported these findings. In conclusion, stevioside, especially at 450 mg, positively impacted growth, biochemical parameters, antioxidant activity, immune response, and gene expression in L. ramada exposed to lead, suggesting its potential to mitigate lead toxicity in aquaculture. Additional research is warranted to investigate the long-term impacts of stevioside supplementation and its prospective implementation in aquaculture.

The modulation of autophagy and unfolded protein response by ent-kaurenoid derivative CPUK02 in human colorectal cancer cells.

BACKGROUND: CPUK02 (15-Oxosteviol benzyl ester) is a semi-synthetic derivative of stevioside known for its anticancer effects. It has been reported that the natural compound of stevioside and its associated derivatives enhances the sensitivity of cancer cells to conventional anti-cancer agents by inducing endoplasmic reticulum (ER) stress. In response to ER stress, autophagy and unfolded protein responses (UPR) are activated to restore cellular homeostasis. Consequently, the primary aim of this study is to investigate the impact of CPUK02 treatment on UPR and autophagy markers in two colorectal cancer cell lines. METHODS: HCT116 and SW480 cell lines were treated with various concentrations of CPUK02 for 72 h. The expression levels of several proteins and enzymes were evaluated to investigate the influence of CPUK02 on autophagy and UPR pathways. These include glucose-regulated protein 78 (GRP78), Inositol-requiring enzyme 1-α (IRE1-α), spliced X-box binding protein 1 (XBP-1 s), protein kinase R-like ER kinase (PERK), C/EBP homologous protein (CHOP), Beclin-1, P62 and Microtubule-associated protein 1 light chain 3 alpha (LC3ßII). The evaluation was conducted using western blotting and quantitative real-time PCR techniques. RESULTS: The results obtained indicate that the treatment with CPUK02 reduced the expression of UPR markers, including GRP78 and IRE1-α at protein levels and XBP-1 s, PERK, and CHOP at mRNA levels in both HCT116 and SW480 cell lines. Furthermore, CPUK02 also influenced autophagy by decreasing Beclin-1 and increasing P62 and LC3ßII at mRNA levels in both HCT116 and SW480 treated cells. CONCLUSIONS: The study findings suggest CPUK02 may exert its cytotoxic effects by inhibiting UPR and autophagy flux in colorectal cancer cells.

Influence of the Nitrogen Fertilization on the Yield, Biometric Characteristics and Chemical Composition of Stevia rebaudiana Bertoni Grown in Poland.

Stevia rebaudiana Bertoni is a plant native to South America that has gathered much interest in recent decades thanks to diterpene glycosides, called steviosides, which it produces. These compounds are characterised by their sweetness, which is 250-300 times higher than saccharose, and they contain almost no caloric value. Stevia is currently also grown outside the South American continent, in various countries characterised by warm weather. This research aimed to determine whether it is viable to grow Stevia rebaudiana plants in Poland, a country characterised by a cooler climate than the native regions for stevia plants. Additionally, the impact of adding various dosages and forms of nitrogen fertiliser was analysed. It was determined that Stevia rebaudiana grown in Poland is characterised by a rather low concentration of steviosides, although proper nitrogen fertilisation can improve various characteristics of the grown plants. The addition of 100 kg or 150 kg of nitrogen per hectare of the field in the form of urea or ammonium nitrate increased the yield of the stevia plants. The stevioside content can be increased by applying fertilisation using 100 kg or 150 kg of nitrogen per hectare in the form of ammonium sulfate. The total yield of the stevia plants grown in Poland was lower than the yield typically recorded in warmer countries, and the low concentration of steviosides in the plant suggests that more research about growing Stevia rebaudiana in Poland would be needed to develop profitable methods of stevia cultivation.

Effect of steviol glycosides as natural sweeteners on glucose metabolism in adult participants.

Steviol glycosides (SGs) are recognized as safe natural sweeteners; however, evidence from randomized controlled trials (RCTs) showed an inconclusive effect of SGs on glucose metabolism in adult participants. We aimed to conduct a systematic review and meta-analysis of RCTs to assess the effect of SGs on glucose metabolism. We systematically searched PubMed, Web of Science and EMBASE to include eligible RCTs. Our primary outcomes were differences between SGs and the control group with respect to changes in blood glucose from the baseline to the end of intervention (including fasting blood glucose [FBG], and HbA1c measurements). A random-effects meta-analysis was conducted for data synthesis to calculate the pooled mean difference (MD). There were twelve RCTs included for analyses with a total of 871 participants (48% females). A significant effect of SGs on FBG (MD = -4.10 mg dl-1, 95% CI -6.55 to -1.65) was found, while no significant difference in HbA1c (MD = 0.01%, 95% CI -0.12% to 0.13%) was observed between SGs and controls. The whole quality of evidence was rated as low. Subgroup analyses demonstrated favorable effects of SGs on FBG in participants aged ≤50 years, those without diabetes mellitus (DM) or hypertension at the baseline, and overweight and obese adults. Sensitivity analyses yielded results largely similar to the main findings. To conclude, SGs are found to produce significant improvement in glucose metabolism in adult participants when compared with the control. More evidence is required to further clarify and support the benefit of SGs as a sugar substitute for glucose metabolism.

Acute and two-week effects of neotame, stevia rebaudioside M and sucrose-sweetened biscuits on postprandial appetite and endocrine response in adults with overweight/obesity-a randomised crossover trial from the SWEET consortium.

BACKGROUND: Sweeteners and sweetness enhancers (S&SE) are used to replace energy yielding sugars and maintain sweet taste in a wide range of products, but controversy exists about their effects on appetite and endocrine responses in reduced or no added sugar solid foods. The aim of the current study was to evaluate the acute (1 day) and repeated (two-week daily) ingestive effects of 2 S&SE vs. sucrose formulations of biscuit with fruit filling on appetite and endocrine responses in adults with overweight and obesity. METHODS: In a randomised crossover trial, 53 healthy adults (33 female, 20 male) with overweight/obesity in England and France consumed biscuits with fruit filling containing 1) sucrose, or reformulated with either 2) Stevia Rebaudioside M (StRebM) or 3) Neotame daily during three, two-week intervention periods with a two-week washout. The primary outcome was composite appetite score defined as [desire to eat + hunger + (100 - fullness) + prospective consumption]/4. FINDINGS: Each formulation elicited a similar reduction in appetite sensations (3-h postprandial net iAUC). Postprandial insulin (2-h iAUC) was lower after Neotame (95% CI (0.093, 0.166); p < 0.001; d = -0.71) and StRebM (95% CI (0.133, 0.205); p < 0.001; d = -1.01) compared to sucrose, and glucose was lower after StRebM (95% CI (0.023, 0.171); p < 0.05; d = -0.39) but not after Neotame (95% CI (-0.007, 0.145); p = 0.074; d = -0.25) compared to sucrose. There were no differences between S&SE or sucrose formulations on ghrelin, glucagon-like peptide 1 or pancreatic polypeptide iAUCs. No clinically meaningful differences between acute vs. two-weeks of daily consumption were found. INTERPRETATION: In conclusion, biscuits reformulated to replace sugar using StRebM or Neotame showed no differences in appetite or endocrine responses, acutely or after a two-week exposure, but can reduce postprandial insulin and glucose response in adults with overweight or obesity. FUNDING: The present study was funded by the Horizon 2020 program: Sweeteners and sweetness enhancers: Impact on health, obesity, safety and sustainability (acronym: SWEET, grant no: 774293).

Tenuifolin improves learning and memory by regulating long-term potentiation and dendritic structure of hippocampal CA1 area in healthy female mice but not male mice.

Polygala tenuifolia Wild is an ancient traditional Chinese medicine. Its main component, tenuifolin (TEN), has been proven to improve cognitive impairment caused by neurodegenerative diseases and ovariectomy. However, there was hardly any pharmacological research about TEN and its potential gender differences. Considering the reduction of TEN on learning and memory dysfunction in ovariectomized animals, therefore, we focused on the impact of TEN in different mice genders in the current study. Spontaneous alternation behavior (SAB), light-dark discrimination, and Morris water maze (MWM) tests were used to evaluate the mice's learning and memory abilities. The field excitatory postsynaptic potential (fEPSP) of the hippocampal CA1 region was recorded using an electrophysiological method, and the morphology of the dendritic structure was examined using Golgi staining. In the behavioral experiments, TEN improved the correct rate in female mice in the SAB test, the correct rate in the light-dark discrimination test, and the number of crossing platforms in the MWM test. Additionally, TEN reduced the latency of female mice rather than male mice in light-dark discrimination and MWM tests. Moreover, TEN could significantly increase the slope of fEPSP in hippocampal Schaffer-CA1 and enhance the total length and the number of intersections of dendrites in the hippocampal CA1 area in female mice but not in male mice. Collectively, the results of the current study showed that TEN improved learning and memory by regulating long-term potentiation (LTP) and dendritic structure of hippocampal CA1 area in female mice but not in males. These findings would help to explore the improvement mechanism of TEN on cognition and expand the knowledge of the potential therapeutic value of TEN in the treatment of cognitive impairment.